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Voltammetric Detection of Cadaverine with DAO-MWCNT Electrochemical Biosensor

Citation & Publication Details

  • Article: “The Voltammetric Detection of Cadaverine Using a Diamine Oxidase and Multi-Walled Carbon Nanotube Functionalised Electrochemical Biosensor.”

  • Journal: Nanomaterials, 2023, Vol 13, Article 36. DOI: 10.3390/nano13010036.

  • Authors: Mohsin Amin, Badr M. Abdullah, Stephen R. Wylie, Samuel J. Rowley-Neale, Craig E. Banks, Kathryn A. Whitehead.

  • Key dates: Received 5 Oct 2022 → Revised 2 Dec 2022 → Accepted 16 Dec 2022 → Published 22 Dec 2022.

  • License: Creative Commons CC-BY 4.0 (open access).

Biological Background: Cadaverine & Periodontal Disease

  • Cadaverine = biogenic diamine; class: polyamines.

  • Produced by bacterial decarboxylation of lysine; present in eukaryotic + prokaryotic cells.

  • Implicated in inflammatory modulation by disrupting host cell-signalling → sustained inflammation [Refs 1–5].

  • Elevated salivary cadaverine correlates with periodontal disease severity (mild → moderate → severe) and tooth loss.

  • Oxidation by Diamine Oxidase (DAO) yields 5-aminobutanal + H2O2, generating a measurable oxidation peak.

Existing Analytical Techniques & Limitations

  • LC-MS, HPLC (fluorescence with o-phthaldialdehyde derivatisation) widely used for food spoilage / clinical diagnostics.

  • Drawbacks: high cost, trained personnel, long turnaround, off-site labs, complex sample prep.

  • Clinical periodontal assessment still relies on
    • Clinical Attachment Level (CAL)
    • Bleeding on Probing (BOP)
    • Pocket Depth (PD)
    – Qualitative, operator-dependent, retrospective, not real-time.

Rationale for Electrochemical Biosensor

  • Biosensors offer: point-of-care, low-cost, miniaturisation, real-time, minimal sample prep.

  • Electrochemical modes: impedimetric, potentiometric, amperometric; current work = amperometric (CV & DPV).

  • Carbon nanomaterials (graphene, CNTs) increase surface area → higher enzyme loading, faster electron transfer.

  • MWCNT advantages: high conductivity, multiple active sites, ease of chemical functionalisation, improved enzyme orientation.

Screen-Printed Electrode (SPE) Fabrication

  • Substrate: polyester film (Autostat).

  • Inks/layers:
    • Working + counter electrodes: carbon graphitic ink; diameter 3.1\;\text{mm}.
    • Reference: Ag/AgCl printed layer.
    • Dielectric mask to insulate tracks.

  • Printing tool: DEK 248 screen printer.

  • Thermal curing: 60^{\circ}\text{C} for 30 min after each layer.

Multi-Walled Carbon Nanotube (MWCNT) Functionalisation Workflow

  1. Carboxylation

    • Acid mix H2SO4 (7.5\;\text{mL}) + HNO_3 (2.5\;\text{mL}); 2\;\text{mg} MWCNT; sonicate 6\;\text{h} at 80^{\circ}\text{C}.

  2. EDC/NHS Activation (zero-length coupling)

    • Suspend carboxylated MWCNT in MES buffer (50\;\text{mM}, pH 6.5).

    • Add EDC (10\;\text{mg mL}^{-1}, 1.2\;\text{mL}) → 1 h RT.

    • Add NHS (50\;\text{mg mL}^{-1}, 2.2\;\text{mL}) → 1 h at 37^{\circ}\text{C}.

  3. Enzyme Conjugation

    • DAO solution 10\;\text{mg mL}^{-1} in 0.1\;\text{M} phosphate buffer.

    • Incubate 2\;\text{mg} MWCNT/EDC-NHS with DAO 37^{\circ}\text{C}, 1 h, 200\;\text{rpm}.

  4. Glutaraldehyde (GA) Crosslinking

    • Add 1\;\text{mL} of 0.2\% GA, shake 30 min RT, then overnight 4^{\circ}\text{C}.

  5. Washing & Storage

    • Tris buffer (100\;\text{mM}, pH 7.2) rinse; store in 0.1\;\text{M} MES at 4^{\circ}\text{C}.

Electrode Functionalisation

  • Drop-cast 10\;\mu\text{L} homogeneous C-MWCNT/DAO/EDC-NHS/GA suspension on SPE working electrode; dry 1 h.

  • Store each SPE in 1\;\text{mL} MES until use.

Physico-Chemical Characterisation

Fourier Transform Infra-Red (FTIR)

  • Pristine MWCNT: characteristic C=C stretch at \sim1600\;\text{cm}^{-1}.

  • Modified MWCNT peaks:
    • Carbonyl C=O stretch \sim3500\;\text{cm}^{-1} (weak).
    • C{-}H stretch \sim2800\;\text{cm}^{-1}.
    • C{=}NH^+ 2363\;\text{cm}^{-1}.
    • C{-}N 1100\;\text{cm}^{-1}.
    • Carboxyl O–H 1715\;\text{cm}^{-1}, C–O 1300\;\text{cm}^{-1}.
    → Confirms DAO + linker attachment.

Energy Dispersive X-ray (EDX)

  • Unmodified surface: C, O, Na, P, S.

  • After modification: new N, Si, Cl signals (from DAO, EDC, NHS).

Electrochemical Measurement Conditions

  • Potentiostat: EmStat3 (PalmSens); software PS-Trace 5.8.

  • Cell: 3-electrode with nickel wire counter, on-chip Ag/AgCl reference.

  • Supporting electrolyte: 0.1\;\text{M} KCl + Britton–Robinson buffer (pH 6 unless stated).

  • Cyclic Voltammetry (CV): potential window [-0.5,\; +1.0]\;\text{V}, scan rates 5–500\;\text{mV s}^{-1}.

  • Differential Pulse Voltammetry (DPV): window [-0.3,\; +0.7]\;\text{V}.

Electrochemical Results & Interpretation

Unmodified SPE

  • At cadaverine 30\;\mu\text{g mL}^{-1} → no discernible redox peaks → molecule electrochemically silent without catalyst.

Modified C-MWCNT/DAO/EDC-NHS/GA SPE

  • Clear redox pair:
    • Anodic (cadaverine oxidation via DAO → 5-aminobutanal + H2O2).
    • Cathodic (reduction of generated H2O2).

  • Scan-rate study:
    • Plot I_p vs \sqrt{\text{scan rate}} linear ( R^2=0.91 ).
    • log–log plot slope 0.29 ≈ theoretical 0.5 for diffusion-controlled, non-porous interface.

Calibration & Analytical Figures

  • DPV used for quantitation.

  • Concentration range tested: 3 – 150\;\mu\text{g mL}^{-1}.

  • Peak current window: \sim32.2 – 43.1\;\mu\text{A} (reported also 140 – 204\;\mu\text{A} due to scaling differences).

  • Dual linear segments observed (low vs high region) → overall linearity maintained.

  • Limit of Blank (LOB): background response with no analyte.

  • Limit of Detection (LOD): \text{LOD}=\text{LOB}+3\sigma_{\text{blank}} \Rightarrow 0.8\;\mu\text{g mL}^{-1}.

  • Sensitivity superior to previous MAO-based biosensor (LOD 19.9\;\mu\text{M}) and laser-scribed graphene device (LOD 50\;\mu\text{M}).

pH Dependence

  • Tested pH 2–12.

  • Oxidation peak potential E_{pa} increases linearly with pH up to pKa of cadaverine (10.25 at 25^{\circ}\text{C}); beyond this, deviation observed.

  • DAO activity & electron transfer thus strongly pH-linked; optimal near physiological pH 6–7.

Performance in Artificial Saliva (Proof-of-Concept TRL Upgrade)

  • Composition per Pytko-Polonczyk et al. [49].

  • At cadaverine 30\;\mu\text{g mL}^{-1} DPV shows:
    • Narrower potential spread.
    • Comparable peak current → negligible interference from salivary constituents.

  • Confirms viability for non-invasive oral diagnostics.

Comparative Table (Literature vs This Work)

  • Meat/fish spoilage sensor: LOD 3\;\mu\text{g kg}^{-1} (chromatographic) – [33].

  • Disposable MAO SPE (cadaverine + putrescine): LOD 9.9/19.9\;\mu\text{M} – [32].

  • Present C-MWCNT/DAO SPE: LOD 0.8\;\mu\text{g mL}^{-1} (≈ 7.8\;\mu\text{M}) – highest sensitivity among electrochemical formats to date.

Advantages, Implications & Future Directions

  • Rapid (~minutes), low-cost, room-temperature, no derivatisation.

  • Drop-cast fabrication easily scalable; compatible with roll-to-roll printing.

  • Enzyme layer can be substituted to target other biogenic amines or metabolites → modular platform.

  • Potential deployment as chair-side periodontal status indicator or food-spoilage tester.

  • Further work: long-term stability, real human saliva trials, multiplexing, wireless data acquisition, integration into microfluidics.

Experimental & Statistical Notes

  • All reagents analytical grade from Merck (Sigma-Aldrich).

  • Water: Type 1, resistivity 18.2\;\text{M}\Omega\,\text{cm}, N$_2$‐degassed.

  • Statistics: GraphPad Prism 9; unpaired t-tests and 1/2-way ANOVA; significance threshold p<0.05.

Ethical, Philosophical & Practical Considerations

  • Provides objective, quantitative periodontal monitoring, reducing clinician bias.

  • Promotes preventative dentistry; early intervention → lower healthcare costs.

  • Open-access publication ensures reproducibility and widespread adoption.

  • Sustainable manufacturing (screen-printing, minimal precious metals) aligns with green chemistry goals.